Ian Adatia, Susan E Barrow, Paula Stratton, James M Ritter, Sheila G Haworth. Abstract ..... Foundation. 1 Haworth SG, Macartney FJ. Growth and development ...
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Br Heart 7 1993;69:179-182
PAEDIATRIC CARDIOLOGY
Abnormalities in the biosynthesis of thromboxane A2 and prostacyclin in children with cyanotic congenital heart disease Ian Adatia, Susan E Barrow, Paula Stratton, James M Ritter, Sheila G Haworth Abstract
Department of Developmental Vascular Biology and Pharmacology, Institute of Child Health, London I Adatia S G Haworth Department of Clinical Pharmacology, United Medical Schools of Guy's and St Thomas' Hospitals, Guy's Hospital Campus, London S E Barrow P Stratton J M Ritter Correspondence to: Professor Sheila G Haworth, Institute of Child Health, 30 Guildford Street, London WC1N 1EH Accepted for publication 10 Au'gust 1992.
between the biosynthesis of thromboxane and
Background-Children with cyanotic the biosynthesis of prostacyclin has been implicongenital heart disease and pulmonary cated in several vascular disorders"1-13 and outflow tract obstruction have shortened might contribute to the pathophysiology of the platelet survival times and are susceptible complications of cyanotic congenital heart to thrombosis and organ infarction. disease with an inadequate pulmonary blood Thromboxane A2 and prostacyclin have flow. The association of a reduced flow and opposing actions on platelet aggregability activated platelets could lead to a disordered and an imbalance in their biosynthesis interaction between the endothelium and might contribute to the pathophysiology platelets. A non-invasive approach to studying thromof these complications. Methods-Biosynthesis of thromboxane boxane and prostacyclin biosynthesis in vivo is A2 and prostacyclin was investigated in 16 to measure excretion of the metabolites children (4-32 months, median 18 2,3-dinor-thromboxane B2 and 2,3-dinormonths) with cyanotic congenital heart 6-oxo-prostaglandin F la'1 15 These metabodisease and pulmonary outflow tract lites reflect extrarenal systemic biosynthesis of obstruction and compared with 16 healthy thromboxane A2 and prostacyclin respecchildren of a similar age (6-34 months, tively.6 17 This method avoids the problem of median 24 months). Urinary excretion of artefactual stimulation of eicosanoid biosyn2,3-dinor-thromboxane B2 (a metabolite thesis that can occur as a result of endothelial of thromboxane A2) and of 2,3-dinor- trauma and platelet activation during blood 6-oxo-prostaglandin Fia (a metibolite of sampling.'8-20 We therefore studied eicosanoid biosynthesis in children with cyanotic congeniprostacyclin) was measured. Results-The children with cyanotic tal heart disease and pulmonary outflow tract congenital heart disease and pulmonary obstruction by measuring the 12 hour urinary outflow tract obstruction excreted more excretion of 2,3-dinor-thromboxane B2 and 2,3-dinor-thromboxane B2 than the 2,3-dinor-6-oxo-prostaglandin F a' healthy children: 916(163) compared with 592(122) nglg creatinine (mean(SEM); 2p = 0.014). The ratio of excretion of Patients and methods 2, 3-dinor-thromboxane B2 to 2, 3-dinor- We studied 16 children (aged 4-32 months, prostaglandin Fla was greater in the median 18 months) with cyanotic congenital patients than in the healthy control group heart disease caused by right ventricular outflow tract obstruction (table). All children had (2.38(0.28) v 1.3(0.22)) (2p = 0.002). Conclusion-The balance between bio- a systemic blood pressure of < 110/70 mm Hg synthesis of prostacyclin and of throm- measured by cuff sphygmomanometry. Twelveboxane A2 is abnormal in children with hour bagged urine specimens were collected cyanotic congenital heart disease and pul- during the hospital admission the night before monary outflow tract obstruction and cardiac catheterisation or operation. A control favours platelet aggregation and vaso- group consisting of 16 healthy normotensive control children (nine male and seven female constriction. aged 6-34 months, median 24 months) was recruited from the Great Ormond Street Hospital staff creche and 12-hour overnight Children with cyanotic congenital heart dis- bagged urine samples were collected similarly. ease and a low pulmonary blood flow owing to Parents of all patients and controls gave right ventricular outflow tract obstruction may informed consent. No patient or control subhave structurally abnormal hypoplastic pulmo- ject took aspirin or other non-steroidal anti nary vessels,' 2 decreased platelet survival inflammatory drug in the two weeks before times,3' and rheological abnormalities render- the study and all urine samples were found ing them susceptible to thrombosis and organ to be free of salicyluric acid.2' Concentrations infarction.6-8 Thromboxane A2 (a vasocon- of 2,3-dinor-6-oxo-prostaglandin Fl. and strictor and promoter of platelet aggregation) 2,3-dinor-thromboxane B2 in the urine were and prostacyclin (a vasodilator and inhibitor measured by immunoaffinity chromatography of platelet aggregation) are derivatives of ara- and gas chromatography mass spectrometry as chidonic acid metabolism.9-" An imbalance described elsewhere.22 Briefly, a well mixed (Br Heart J 1993;69:179-182)
Adatia, Barrow, Stratton, Ritter, Haworth
180
Clinical observations Case no
Sex
Age (mth) Diagnosis
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Median
M M M M F M M M M F F F F F M M -
4 5 7 8 11 11 11 18 18 21 22 24 25 26 28 32 18
PAT, VSD TOF TOF VSD,PA band TOF TOF TGA,VSD, PS TOF TOF PAT, IVS, RBT PAT,VSD, RBT TA, PS,VSD, LBT TOF,RBT TOF, RBT TGA,VSD, PS, RBT TOF, R&LBT -
Qp:Qs
(%)
Platelets (x 1hfmm3)
Hb (gldl)
Drugs
BP (mm Hg)
NA NA 09 07 07 04 NA 0-4 0-8 0-6 0-6 05 05 NA 0-7 0-2 0-6
75 81 86 84 70 71 78 75 85 84 75 74 76 65 70 45 75
259 696 501 563 367 170 223 360 264 236 144 235 311 253 250 48 256
17-6 14-7 12 7 12-3 14-4 19-3 12-7 16-6 14-4 13-4 19-3 16 14-3 14-8 19-6 17-5 14-8
Nil Propranolol Propranolol Nil Propranolol Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil -
90/50 80/45 80/50 95/60 102/60 100/65 95/50 105/65 104/75 95/50 100/75 102/50 104/47 85/50 110/60 100/60 100/55
AO Sat
TOF, Tetralogy of Fallot; VSD, ventricular septal defect; IVS, intact ventricular septum; R/LBTS, right/left modified BlalockTaussig shunt; PA, pulmonary artery; PAT, pulmonary atresia; PS, pulmonary stenosis; TGA, transposition of the great arteries; AO Sat, oxygen saturation; Hb, haemoglobin; Qp:Qs, pulmonary to systemic flow ratio; NA, not available.
sample of 30-50 ml urine was stored at - 20°C until assay. Urine samples (10 ml) were diluted 1:1 by volume with buffer at pH 8-0 and [2H4] 2,3-dinor-thromboxane B2 and [2H4] 2,3-dinor-6-oxo-prostaglandin Fla (5 ngl each) were added. Eicosanoids were extracted on cyanogen bromide-activated sepharose columns containing immobilised antibodies that had been raised against 6-oxo-prostaglandin Fla and thromboxane B2 and that crossreacted with their respective 2,3-dinor metabolites. Urine samples were applied under vacuum to the columns, which were washed with water (10 ml). Eicosanoids were eluted by addition of 2 x 0X5 ml acetone:water (95:5) and rotation of the columns for 15 min. Samples were taken to dryness (N2 stream) and were derivatised as 3,5-bis-trifluoromethylbenzyl esters and trimethylsilyl ethers.23 They were analysed by a VG 70-SEQ gas chromatograph/mass spectrometer in the electron capture mode with methane or ammonia as the reagent gas. Carboxylate anions at mass/charge (m/z) ratio of 557 were monitored for 2,3-dinor-6-oxo-prostaglandin Fla and 2,3-dinor-thromboxane B2 and at m/z 561 for the deuterated internal standards. The detection limit for each eicosanoid was 5 pg/ml. Urinary creatinine concentrations were measured by standard methods. Results are expressed as mean (SEM) ng eicosanoid per g creatinine. The Mann-Whitney U test was used in statistical analysis and differences were regarded as significant when 2p < 0 05.
than in the healthy children (1-3 (0 22)) (2p = 0 002) (fig 3). Among the cyanotic children there was no correlation between excretion of 2,3-dinor-thromboxane B2 and 2,3-dinor-6-oxo-prostaglandin Fla or their ratio and the platelet count, haemoglobin, or pulmonary to systemic flow ratio (Qp:Qs). Discussion We showed an increase in the urinary excretion of 2,3-dinor-thromboxane B2 and in the ratio of urinary 2,3-dinor-thromboxane B2 to 1200-
X,. c
2p
=
0.014
800-
.c ._p V CU 600CD) _
-S
200-
D
0-
Low Qp v controls Figure 1 Excretion of 2,3-dinor-thromboxane B2 (nglg creatinine) in children with a low pulmonary blood flow (Qp) and in controls. TX thromboxane.
800 a
U.
Results The patients with cyanotic congenital heart disease excreted significantly more 2,3-dinorthromboxane B2 than the control group: 916(163) compared with 592(122) ng/g creatinine (2p = 0-014) (fig 1). Excretion of
-T-
1000
m
700-
a 0L 6x (D C: 1 1.
600-
2p
=
0-08 (NS)
7-
5o
-500
D 400-
-60 m 300 C;) 200-
D: 2,3-dinor-6-oxo-prostaglandin Fl5l was 381 100. .S with (61) in the cyanotic children compared 589 (95) ng/g creatinine in the controls (2p = 0 08) (fig 2). The ratio of 2,3-dinorLow Qp v controls thromboxane B2 to 2,3-dinor-6-oxo-prosta- Figure 2 Excretion of 2,3-dinor-6-oxo-prostaglandin F,,, glandin F1I was significantly greater in (nglg creatinine) in children with a low pulmonary blood the patients with heart disease (2-38 (0-28)) flow (Qp) and in controls. PG, prostaglandin. 0-
181
Thromboxane A2 and prostacyclin in cyanotic congenital heart disease 3-1-
2p
=
0002
0cL
x 0 ._
cc
1-
sion.26 This is an acute disease, however, in which compensatory mechanisms may not be effective, unlike the slower progression of hypoxaemia caused by worsening right ventricular outflow tract obstruction in congenital heart malformations. Although increased thromboxane biosynthesis is probably due mainly to platelet dysfunction, in vitro studies suggest that the exposure of the endothelium to reduced oxygen tension might also be
relevant.2'-" 0-
Low Qp v contr ols Figure 3 Ratio of 2,3-dinor-thromboxa? ,ze B2 to 2,3-dinor-6-oxo-prostaglandin F,, in chilfdren with a low pulmonary blood flow (Qp) and in contr(ols. TX, thromboxane; PG, prostaglandin.
2,3-dinor-6-oxo-prostaglandin FIA in young children with cyanotic congenital heart disease and pulmonary outflow tract obstruction. The main source of thromboxane A2 excreted as 2,3-dinor-thromboxane B2 is thought to be activated platelets though this eicosanoid may also be produced by endothelial cells and macrophages.24 25 It seems likely that in cyanotic congenital heart disease, increased excretion of 2,3-dinor-thromboxane B2 is primarily of platelet origin as there is evidence of abnormal platelet function in such children.7 Four of our patients had abnormal platelet counts (table 1). In three patients with mild systemic arterial oxygen desaturation the counts were high (> 500 000 per mm3). In one patient, with the lowest arterial oxygen saturation, the platelet count was low (48 000 per mm3). Though the patient with the lowest platelet count excreted the most 2,3-dinorthromboxane B2, in the group as a whole there was no correlation between 2,3-dinor-thromboxane B2 and the platelet count. Thrombocytopenia is a late event in cyanotic congenital heart disease. It usually accompanies severe hypoxaemia and polycythaemia.3 With the contemporary practice of early palliation or corrective surgery thrombocytopenia is now an uncommon finding in young patients. Platelet half times are, however, known to be reduced in patients who have cyanotic congenital heart disease with mild to moderate arterial oxygen desaturation but there is a compensatory increase in platelet production that maintains platelet numbers in the normal or higher than normal range.34 The increased biosynthesis of thromboxane A2in the patients in this study, most of whom had normal platelet counts, could therefore reflect increased platelet activation with a compensatory rise in thrombocyte production. An increase in platelet activation may also explain the paradoxical clinical observation that thrombotic episodes are seen more commonly in children under two years of age before the development of severe polycythaemia and thrombocytopenia.7 A negative correlation between 2,3-dinor-thromboxane B2 and platelet count has been reported previously in women with pregnancy-induced hyperten-
We conclude therefore that in children with cyanotic congenital heart disease and a low pulmonary blood flow there is an increase in thromboxane A2 biosynthesis and in ratio of thromboxane A2 to prostacyclin. We suggest that these abnormalities precede the development of severe polycythaemia and thrombocytopenia. The increase in the ratio of thromboxane A2 to prostacyclin which favours vasoconstriction and platelet aggregation may contribute to the development of thrombotic episodes in such children. The findings also support the current practice of early correction, rather than palliation, because even mild to moderate systemic desaturation seems to promote the biosynthesis of thromboxane A2V The presence of abnormally high thromboxane A2 production provides a therapeutic rationale for the widely used practice of prescribing aspirin to maintain the patency of systemic to pulmonary artery shunts in those children with unfavourable anatomy who require palliation as an initial procedure. We thank Professor A I Mallet (Director, UMDS Mass Spectrometry Unit, St Thomas' Campus) for use of mass spectrometry facilities, Dr R Ersser for salicyluric acid analysis, Ms Janet Kipping (Creche matron), the nursing staff of Richard Bonham Carter ward, Great Ormond Street Hospital for help with the urine collections, and Miss Mary Shearer for secretarial assistance. This work was supported by the British Heart Foundation. 1 Haworth SG, Macartney FJ. Growth and development of pulmonary circulation in pulmonary atresia with ventricular septal defect and major aortopulmonary collateral arteries. Br Heart J 1980;44: 14-24. 2 Johnson RJ, Haworth SG. Pulmonary vascular and alveolar development in tetralogy of Fallot: a recommendation for early correction. Thorax 1982;37:893-901. 3 Gross S, Keefer V, Liebman J. The platelets in cyanotic congenital heart disease. Pediatrics 1968;42:651-8. 4 Waldman JD, Czapek EE, Paul MH, Schwartz AD, Levin DL, Schindler S. Shortened platelet survival in cyanotic heart disease. Jf Pediatr 1975;87:77-9. 5 Ihenacho HN, Breeze GR, Fletcher DJ, Stuart J. Consumption coagulopathy in congenital heart disease. Lancet
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